Summary: HIV envelope glycoprotein gp120 contains epitopes which are targeted by broadly cross-reactive neutralizing antibodies in humans and which depend on the native protein conformation. Monoclonal antibodies to these sites can neutralize a broad range of HIV-1 isolates and protect monkeys against viral challenge. Based on mapping these epitopes on the three dimensional structure of gp120, we have found an acceptor site, where foreign protein sequences can be inserted without disrupting the native folding of gp120. By inserting a potent immunogen at this site, we could enhance the intrinsic vaccine potency of gp120, while retaining important conformational sites needed to elicit these antibodies. Two types of carrier protein have been inserted at this site, hepatitis B surface antigen (HBsAg) and hepatitis B core antigen. The resulting protein hybrids should combine the immunogenicity of the carrier protein with the neutralizing epitopes of gp120. Hybrid proteins were expressed in a recombinant viral vector and showed normal glycosylation, dual antigenicity, and high affinity binding of CD4,indicating native folding at the CD4 binding site. Although native core antigen forms multimeric particles, few of the core/gp120 hybrids were able to assemble. In contrast, surface antigen/gp120 hybrids assembled particles with high efficiency. These particles showed dual antigenicity and native folding of gp120. They bound CD4-Ig and a panel of broadly reactive human neutralizing monoclonals nearly as well as native gp120. They banded in CsCl at a density indicating a lipid content similar to native HBsAg particles. EM shows 22 nm particles, similar to surface antigen alone. By analogy with HBsAg, these particles diplay an array of about 200 gp120 molecules at the lipid/water interface, similar to the surface of HIV virions. Assembly of gp120 into virus-like particles has been a long-term goal of this laboratory. The particles' immunogenicity will be tested in mice and monkeys. In the case of other successful vaccines, such as HBsAg vaccine for hepatitis B virus, and L1 capsid protein of human papilloma virus, multimers were up to 1,000-fold more potent than the same weight of monomers. This is the first multimeric gp120 vaccine, and we anticipate that it may elicit a strong immune response in mice and monkeys. High responder monkeys would be challenged with titered stocks of SHIV challenge virus. Through potent carrier and multimer effects, this construct may have a profound effect on HIV immunology. This project is funded by an intramural NIH grant.